P1425: SYSTEMATIC INVESTIGATION OF HYDROXYUREA TREATMENT EFFECT IN PEDIATRIC PATIENTS WITH SICKLE CELL DISEASE
Authors: Varsha Bhat, Alka A. Potdar, Greg Gibson, G. Karen Yu, Vivien Sheehan
Journal: HemaSphere (2023)

Topic: 26. Sickle cell disease Background: Sickle cell disease (SCD) is a severe, inherited, multisystem disorder that affects millions of people globally. Hydroxyurea (HU), a disease-modifying therapy approved for pediatric and adult patients with SCD, increases fetal hemoglobin (HbF) levels, which leads to decreased polymerization of deoxygenated sickle hemoglobin. However, the exact mechanisms underlying the effect of HU on SCD pathophysiology and the reasons for interindividual variability in clinical response remain unclear. Here, we systematically investigate response to HU, using a unique cohort of pediatric patients treated to the maximum tolerated dose (MTD) of HU that includes matched baseline samples and HU washout samples. Aims: To assess the impact of HU on hematologic laboratory measurements and whole blood transcriptomes of pediatric patients with SCD. Methods: Informed consent was obtained for all patients. Whole-blood samples from patients with SCD were collected before initiating HU (age <21 years) and at HU MTD (targeted near an absolute neutrophil count [ANC] ≤2000 cells/µL or an absolute reticulocyte count [ARC] ≤70 cells/µL; age ≤21 years). HU washout samples were collected ≥3 months after HU discontinuation when red blood cell (RBC) indices had returned to pre-HU levels. Linear mixed models were used to determine the impact of HU treatment on key laboratory measurements using paired samples. RNA-Seq was carried out with a 150 base pair, paired-end protocol on a HiSeq 4000 instrument. Differential gene expression and biological pathway enrichment analyses of the pre- and post-HU treatment samples were carried out using DESeq2 and gene set enrichment analysis. Results: Demographics and lab measurements of the 25 patients included in the study are presented in the Table. All patients reached their MTD on HU therapy within 10 months. Compared with baseline, there was a robust increase in %HbF levels at MTD which was accompanied by a significant increase in hemoglobin (Hb) and mean corpuscular volume (MCV) and a significant decrease in white blood cell count (WBC), ANC, and ARC (Table). Following HU washout (n=2), the %HbF, MCV, and ARC returned to levels comparable to those at baseline. However, ANC (per μL) remained comparable to HU MTD (HU MTD: patient 1=3700, patient 2=4290; HU washout: patient 1=4480, patient 2=4420). Analyses of the RNA-Seq data revealed that HU downregulated genes associated with heme metabolism, oxidative phosphorylation, reactive oxygen species, and interferon alpha response pathways and upregulated genes associated with inflammatory response, TNFα signaling, and IL2/STAT5 signaling pathways. In addition, we observed a strong positive correlation between the transcript-per-million counts of the HBG2 gene and HbF levels (Pearson’s r: 0.93, P<2.2e–16). Several patients showed increased %HbF by HU without a reduction in the gene expression of known regulators of HbF levels, BCL11A and/or ZBTB7A. An effect of age was seen on %HbF and on expression of multiple genes; further analysis is underway to adjust for a potential confounding effect. Summary/Conclusion: HU alters transcription in favorable ways outside of HbF induction. Genes involved in heme metabolism, which includes both heme synthesis and degradation, were downregulated with HU treatment, consistent with elevated %HbF and total Hb and a reduction in ARC due to reduced RBC turnover. Further examination of baseline and HU MTD transcriptomes may add to our understanding of the HbF-related and non-related effect of HU on blood cells. Funding: Pfizer Inc.Keywords: Sickle cell disease, RNA-seq, Hydroxyurea